Sepsis is morbid condition frequently induced by a toxin, the introduction or accumulation of which is most commonly caused by infection or trauma. The initial symptoms of sepsis or septic shock typically include chills, profuse sweat, irregularly remittent fever, prostration and the like, followed by persistent fever, hypotension leading to shock, neutropenia, leukopenia, disseminated intravascular coagulation, adult respiratory distress syndrome and multiple organ failure. These final symptoms, generally referred to as acute phase septic shock, almost invariably lead to death.
Sepsis-inducing toxins have been found associated with pathogenic bacteria, viruses, plants and venoms. Among the well described bacterial toxins are the endotoxins or lipopolysaccharides (LPS) of the gram-negative bacteria. These molecules are glycolipids that are ubiquitous in the outer membrane of all gram-negative bacteria. While the chemical structure of most of the LPS molecule is complex and diverse, a common feature is the lipid A region of LPS (Rietschel et al., 1984, In Handbook of Endotoxins, eds. R. A. Proctor and E. Th. Rietschel, Elsevier, Amsterdam 1:187-214). Recognition of lipid A in biologic systems initiates many, if not all, of the pathophysiologic changes of sepsis. Because lipid A structure is highly conserved among all types of gram-negative organisms, common pathophysiologic changes characterize gram-negative sepsis.
Current concepts support the contention that the primary response of the host (including man) to LPS involves the recognition of LPS by cells of the monocyte/macrophage lineage, followed by the rapid elaboration of a variety of cell products including the general group known as cytokines. Other cell types believed to participate in sepsis and in particular in the response to LPS are polymorphonuclear leukocytes and endothelial cells; each of these cell types are also capable of responding to LPS with the elaboration of potent inflammatory substances.
LPS is believed to be a primary cause of death in humans during gram-negative sepsis, particularly when the symptoms include adult respiratory distress syndrome (ARDS) (van Deventeret al., 1988, Lancet, 1:605: Ziegler et al., 1987, J. Infect. Dis. 136:19-28). For instance, one particular cytokine, tumor necrosis factor alpha/cachectin (TNF), has been reported to be a primary mediator of septic shock (Beutler et al., 1987, N. Eng. J. Med. 316:379). Intravenous injection of LPS endotoxin from bacteria into experimental animals and man produces a rapid, transient release of TNF (Beutler et al., 1985, J. Immunol. 135:3972; Mathison et al., 1988, J. Clin. Invest. 81:1925). Evidence that TNF is a critical mediator of septic shock comes primarily from experiments in which pretreatment of animals with anti-TNF antibodies reduces lethality (Beutler et al., 1985, Science 229:869; Mathison et al., supra). These reports suggest that interruption of the secretion of TNF caused by LPS or other factors would ameliorate the often lethal symptoms of sepsis.
Upon introduction of LPS into the blood, it may bind to a protein termed lipopolysaccharide binding protein (LBP). LBP is a 60 kD glycoprotein present at concentrations of less than about 5 .mu.g/ml in the serum of healthy animals and man. During the acute phase, LBP is synthesized by hepatocytes, and reaches concentrations of 30-50 .mu.g/ml in serum. LBP can be purified from acute phase human and rabbit serum (Tobias et al., 1986, J. Exp. Med. 164:777-793). LBP recognizes the lipid A region of LPS and forms complexes with both rough and smooth form LPS (Tobias et al., 1989, J. Biol. Chem. 264:10867-10871). LBP bears N-terminal sequence homology with the LPS-binding protein known as bactericidal permeability-increasing factor, (BPI) (Tobias et al., 1988, J. Biol. Chem. 263:13479-13481). BPI is stored in the specific granules of PMN (Weiss et al., 1987, Blood 69:652-659) and kills gram-negative bacteria by binding LPS and disrupting the permeability barrier (Weiss et al., 1984, J. Immunol. 132:3109-3115).
In contrast to BPI, LBP is not directly cytotoxic for gram-negative bacteria (Tobias et al., 1988, J. Biol. Chem. 263:13479-13481). Instead, LBP binding to LPS has been found to dramatically enhance the interaction of LPS with macrophages, indicating its role as an opsonin (Wright et al., 1989, J. Exp. Med. 170:1231-1241). Inhibition of LBP, e.g., with an anti-LBP antibody, has been suggested as therapeutically useful for treating endotoxin-mediated sepsis (International Patent Application No. PCT/US90/04250, filed Jul. 30, 1990).
Studies over the past few years have shown that plasma proteins play an important role in mediating responses of cells to low concentrations of bacterial lipopolysaccharide. LBP (Shumann et al., 1990, Science 249:1429) and Septin (Wright et al., 1992, J. Exp. Med. 176:719) interact with LPS and facilitate the binding of the LPS to CD14 (Hailman et al., 1994, J. Exp. Med. 179:269). CD14, a protein found on the surface of monocytes, macrophages and neutrophils (Goyert & Ferrero, 1987, In Leukocyte Typing III, McMichael et al., eds., Springer Verlag: New York, p. 613) then initiates responses of these cells. CD14 is also found as a soluble protein in the plasma, and complexes of LPS with soluble CD14 participate in responses of endothelial cells (Frey et al., 1992, J. Exp. Med. 176:1665), epithelial cells (Pugin et al., 1993, Proc. Natl. Acad. Sci. USA 90:2744), and probably other cell types that do not express membrane CD14. These studies have emphasized the ability of plasma to potentiate responses to LPS. In short, plasma contains factors that enhance the ability of LPS to initiate cellular activation, and ultimately, septic shock.
In contrast, a number of older studies have focused on the ability of plasma to inactivate endotoxin (Rall et al., 1957, Am. J. Physiol. 188:559; Skarnes et al., 1958, J. Exp. Med. 108:685; Ulevitch et al., 1978, J. Clin. Invest. 62:1313). Incubation of LPS with plasma has been shown to block the ability of the LPS to cause fever and death in experimental animals (Skarnes et al., supra; Ulevitch et al., supra) and to block the ability of LPS to give a positive signal in the Limulus amebocyte lysate (LAL) assay (Johnson et al., 1977, Am. J. Pathology 88:559; Emancipator et al., 1992, Infect. Immunol. 60(2):596). This "detoxification" or neutralization of LPS is thought to occur without covalent modification of the LPS, because the LPS detoxified by plasma can be extracted with organic solvents and shows full activity (Rudbach & Jolnson, 1964, Nature 202:811).
Work from several laboratories has shown that plasma lipoproteins, particularly high-density lipoproteins (HDL), bind and neutralize LPS (Skarnes et al., 1968, J. Bacteriology 95:2031); Ulevitch et al., 1979, J. Clin. Invest. 64:1516; Munford et al., 1981, Infect. Immunol. 34(3):835; Flegel et al., 1993, Infect. Immunol. 61(12):5140) and that these particles may constitute the LPS-neutralizing activity in plasma.
An LPS neutralizing factor was previously identified as a factor that inhibited binding of LPS to monocytes, macrophages, and polymorphonuclear leukocytes; destroyed LPS-coated erythrocyte binding to macrophages, and destroyed septin-dependent stimulation of polymorphonuclear leukocytes and monocytes (see copending U.S. patent application Ser. No. 08/188,644, filed Jan. 27, 1994, which is a continuation of application Ser. No. 07/814,775, filed Dec. 30, 1991, now abandoned, which was a continuation in part of application Ser. No. 07/473,609, filed Feb. 1, 1990, now abandoned, and see International Patent Publication WO 93/13201, published Jul. 8, 1993, by Wright, each of which is incorporated herein by reference in their entireties). The activity of "Septinase," as this factor was termed, could be inhibited by .alpha..sub.2 -macroglobulin. One mechanism by which this factor was believed to inactivate Septin was by proteolysis; other experimental data suggested that the factor inactivated LPS without destroying Septin. Whatever the underlying mechanism, Septinase permanently inactivated LPS.
A number of approaches for treating sepsis have been attempted. These include use of antibodies to LPS, use of antibodies to tumor necrosis factor, use of a soluble TNF receptor, use of a soluble interleukin-1 (IL-1) receptor, to name a few. While each approach has some efficacy, the overall results have been disappointing.
Thus, there is a need in the art for an effective pharmaceutical agent and method of treatment for neutralizing gram-negative endotoxin (i.e., LPS), in order to prevent or alleviate symptoms of sepsis and septic shock.
The citation of any reference herein should not be considered as an admission that such reference is available as prior art to the invention.